The native intermediate filament network within human corneal endothelium was identified by a monoclonal antibody to vimentin intermediate filament protein. Human corneal endothelial cells in tissue culture were shown to react positively to this monoclonal antibody by indirect immunofluorescence microscopy. In cryostat sections of human cornea, only the endothelial cells and keratocytes stained for vimentin. Fluorescent staining patterns of the cultured endothelial cells demonstrated that vimentin forms arrays of cytoplasmic filaments which encapsulate the nucleus and anchor in the apical junctions. These results reveal a previously undescribed cytoskeleton in human corneal endothelium and suggest that the intermediate filament vimentin comprises this structural network which is, in part, responsible for nuclear centration and cell-to-cell contacts. In addition, these results may indicate the possible embryogenesis of the corneal endothelium, since vimentin is a marker for cells of mesenchymal origin.